Physical Sciences Inc. (PSI) and the laboratory of Christopher Gabel at Boston University School of Medicine propose to design, develop, characterize, and demonstrate a novel approach to simultaneous multichannel microscopy that will overcome a number of limitations of existing technologies. Simultaneous multichannel microscopy techniques measure changes in multiple observables that are reported by different wavelengths of light, and these techniques are finding greater application in many areas of biological research as noninvasive measurements become more attainable and desirable. Example applications include in vivo ratiometric neuronal calcium imaging, co-localization of multiple fluorescent proteins and labels, and non-invasive pH measurement. Despite the ubiquity of multichannel imaging and the existence of three current commercial approaches to perform simultaneous multichannel imaging, limitations to each approach prevent full utilization and exploitation of the technique. The long-term objective of this project is to develop a simple, inexpensive commercial device that enables standard single-channel fluorescence microscopes to perform simultaneous multichannel microscopy. This approach can easily scale to higher numbers of channels and reduces photobleaching by simultaneous imaging, advancing the capabilities of fluorescence microscopy. Furthermore, the device contains no moving parts and does not require alignment or specialized expertise to operate, allowing advanced imaging while maintaining user-friendliness. Scientific program areas benefitting directly from the novel approach include molecular imaging, optical imaging and spectroscopy, and tissue engineering. During the Phase I program we propose to fabricate and optically test prototypes of the device to confirm adherence to commercial imaging standards. In particular, prototypes will be constructed for and tested in three locations in conventional microscopes to determine the optimal location and configuration for performance, cost, and convenience. Device performance will be evaluated by imaging standard fluorophores in the nematode worm C. elegans and cell culture and comparing these data to corresponding measurements on conventional multichannel imaging devices. Advanced engineering will be performed in Phase II to reduce device cost, further evaluate it, and develop a path for technology commercialization.